[Show abstract][Hide abstract]ABSTRACT: Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis.

[Show abstract][Hide abstract]ABSTRACT: The multicomponent global regulator Velvet complex has been identified as a key regulator of secondary metabolite production in Aspergillus and Penicillium species. Previous work indicated a massive impact of PcvelA and PclaeA deletions on penicillin production in prolonged batch cultures of P. chrysogenum, as well as substantial changes in transcriptome. The present study investigated the impact of these mutations on product formation and genome-wide transcript profiles under glucose-limited aerobic conditions, relevant for industrial production of β-lactams. Predicted amino acid sequences of PcVelA and PcLaeA in this strain were identical to those in its ancestor Wisconsin54-1255. Controls were performed to rule out transformation-associated loss of penicillin-biosynthesis clusters. The correct PcvelA and PclaeA deletion strains revealed a small reduction of penicillin G productivity relative to the reference strain, which is a much smaller reduction than previously reported for prolonged batch cultures of similar P. chrysogenum mutants. Chemostat-based transcriptome analysis yielded only 23 genes with a consistent differential response in the PcvelAΔ and PclaeAΔ mutants when grown in the absence of the penicillin G side-chain precursor phenylacetic acid. Eleven of these genes belonged to two small gene clusters, one of which contained a gene with high homology to the aristolochene synthase. These results provide a clear caveat that the impact of the Velvet complex on secondary metabolism in filamentous fungi is strongly context dependent.

[Show abstract][Hide abstract]ABSTRACT: Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporin-related products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. chrysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via β-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of β-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of β-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis.

[Show abstract][Hide abstract]ABSTRACT: The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-(13)C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH(4))(2)SO(4) as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate.

[Show abstract][Hide abstract]ABSTRACT: In microbial production of non-catabolic products such as antibiotics a loss of production capacity upon long-term cultivation (for example chemostat), a phenomenon called strain degeneration, is often observed. In this study a systems biology approach, monitoring changes from gene to produced flux, was used to study degeneration of penicillin production in a high producing Penicillium chrysogenum strain during prolonged ethanol-limited chemostat cultivations.
During these cultivations, the biomass specific penicillin production rate decreased more than 10-fold in less than 22 generations. No evidence was obtained for a decrease of the copy number of the penicillin gene cluster, nor a significant down regulation of the expression of the penicillin biosynthesis genes. However, a strong down regulation of the biosynthesis pathway of cysteine, one of the precursors of penicillin, was observed. Furthermore the protein levels of the penicillin pathway enzymes L-α-(δ-aminoadipyl)-L-α-cystenyl-D-α-valine synthetase (ACVS) and isopenicillin-N synthase (IPNS), decreased significantly. Re-cultivation of fully degenerated cells in unlimited batch culture and subsequent C-limited chemostats did only result in a slight recovery of penicillin production.
Our findings indicate that the observed degeneration is attributed to a significant decrease of the levels of the first two enzymes of the penicillin biosynthesis pathway, ACVS and IPNS. This decrease is not caused by genetic instability of the penicillin amplicon, neither by down regulation of the penicillin biosynthesis pathway. Furthermore no indications were obtained for degradation of these enzymes as a result of autophagy. Possible causes for the decreased enzyme levels could be a decrease of the translation efficiency of ACVS and IPNS during degeneration, or the presence of a culture variant impaired in the biosynthesis of functional proteins of these enzymes, which outcompeted the high producing part of the population.

[Show abstract][Hide abstract]ABSTRACT: Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of ß-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA.